Macrophage colony-stimulating factor-, granulocyte-macrophage colony-stimulating factor-, or IL-3-dependent survival of macrophages, but not proliferation, requires the expression of p21(Waf1) through the phosphatidylinositol 3-kinase/Akt pathway

Mouse bone marrow-derived macrophages proliferate in the presence of macrophage colony-stimulating factor (M-CSF), granulocyte-macrophage colony-stimulating factor, or IL-3, but undergo apoptosis in their absence. Inhibition of extracellular signal-regulated kinases (ERK)-1/2 blocks growth factor-dependent proliferation but not survival, indicating that the two processes require independent signaling pathways. Although M-CSF induces the activation of other kinase pathways, such as c-Jun N-terminal kinase, p38, and phosphatidylinositol 3-kinase (PI-3K), these pathways are not required for proliferation. However, PI-3K is the only one necessary for the induction of survival, as demonstrated using the inhibitors LY294002 and Wortmannin. Growth factors also activate Akt kinase and a transient expression of the cdk inhibitor p21(Waf1), which inhibits apoptosis but is not required for proliferation. PI-3K inhibitors also block growth factor-dependent expression of p21(Waf1) and the activation of Akt. Moreover, the survival induced by cyclosporin A or decorin is also dependent on the PI-3K/Akt kinases and p21(Waf1). These findings demonstrate that the induction of p21(Waf1) through the PI-3K/Akt pathway is a general survival response of macrophages. Our results show that growth factors in macrophages use two pathways: one for proliferation, mediated by ERK, and the other for survival, which requires the PI-3K/Akt kinases and p21(Waf1).     

IL‐4 induces protection of vascular endothelial cells against killing by complement and melittin through lipid biosynthesis

We have shown previously that cytokines IL‐4 and IL‐13 induce protection in porcine vascular endothelial cells (EC) against killing by the membrane attack complex (MAC) of human complement. This protection is intrinsic, not due to changes in complement regulatory proteins, and requires activation of Akt and sterol receptor element binding protein‐1 (SREBP‐1), which regulates fatty acid and phospholipid synthesis. Here we report that, compared to EC incubated in medium, IL‐4‐treated EC had a profound reduction in complement‐mediated ATP loss and in killing assessed by vital dye uptake, but only a slight reduction in permeability disruption measured by calcein release. While controls exposed to complement lost mitochondrial membrane potential and subsequently died, protected EC maintained mitochondrial morphology and membrane potential, and remained alive. SREBP‐1 and fatty acid synthase activation were required for protection and fatty acid and phospholipid synthesis, including cardiolipin, were increased after IL‐4 stimulation, without increase in cholesterol content or cell proliferation. IL‐4 also induced protection of EC from killing by the channel forming protein melittin, similar to protection observed for the MAC. We conclude that IL‐4 induced activation of Akt/SREBP‐1/lipid biosynthesis in EC, resulting in protection against MAC and melittin, in association with mitochondrial protection.     

The proangiogenic phenotype of human tumor-derived endothelial cells depends on thrombospondin-1 downregulation via phosphatidylinositol 3-kinase/Akt pathway

umor-derived endothelial cells (TEC) display increased survival and angiogenic properties in respect to normal endothelial cells. The aim of this study was to investigate the mechanism potentially involved in TEC proangiogenic phenotype. We found that thrombospondin-1 (TSP-1), a potent physiological inhibitor of angiogenesis, was significantly reduced in TEC in respect to normal endothelial cells. This reduction was confirmed by immunofluorescence in the intratumor vessels of clear cell renal carcinomas. As TEC were shown to display a basal upregulation of the phosphatidylinositol 3-kinase (PI3K)/Akt pathway, we evaluated the possible regulation of TSP-1 by this pathway by using LY294002 and wortmannin, the PI3K inhibitors, and rapamycin, the mammalian target of rapamycin (mTOR) inhibitor. In addition, we developed negative dominant TEC for Akt. TSP-1 production by TEC was enhanced by the treatment with LY294002 and wortmannin and with rapamycin, suggesting a negative regulation of TSP-1 expression by the PI3K/Akt/mTOR pathway. In addition, downregulation of Akt activation in negative dominant Akt TEC enhanced TSP-1 expression and release. Administration of exogenous TSP-1 to TEC reduced their proangiogenic properties in vitro and in vivo. In parallel, blockade of TSP-1 with an anti-TSP-1 antibody in negative dominant Akt TEC restored their proangiogenic phenotype to levels similar to wild-type TEC. In conclusion, these results indicate that the upregulation of the PI3K/Akt/mTOR pathway is responsible for the inhibition of TSP-1 synthesis which is critical in determining the proangiogenic phenotype of TEC. Strategies aimed to inhibit the PI3K/Akt/mTOR pathway may restore a normal quiescent endothelial phenotype in TEC by promoting TSP-1 production.     

MEK1/2在脂多糖诱导人脐静脉内皮细胞ICAM-1表达中的作用

目的：探讨MEK1/2在脂多糖诱导人脐静脉内皮细胞（HUVECs）ICAM-1表达中的作用。 方法： 用不同浓度LPS或LPS加MEK1/2特异性抑制剂PD98059和HUVECs孵育不同时间后，分别采用RT-PCR和Western blotting检测ICAM-1 mRNA和蛋白的表达。 结果： LPS呈时间-浓度依赖性地上调HUVECs ICAM-1 mRNA和蛋白的表达。LPS预处理后2 h，HUVECs ICAM-1 mRNA和蛋白的表达即开始升高，LPS(100 μg·L-1)作用后6 h,ICAM-1 mRNA和蛋白的表达基本达到高峰；PD98059(10 μg·L-1)可显著抑制LPS(100 μg·L-1)诱导6 h的ICAM-1 mRNA和蛋白的表达，抑制率分别为54.4%和44.9%（P<0.01 vs LPS）。 结论： 调控MEK1/2通路可能为内毒素休克诱导血管内皮损伤的防治提供新的策略。     

P13K/Akt信号通路在白蛋白诱导肾小管上皮细胞转化生长因子β1表达中的作用

目的 研究白蛋白对人肾小管上皮细胞(HK-2)分泌转化生长因子β1(TGF-β1)的影响,以及P13K/Akt信号通路在这一过程中的作用.方法 体外培养HK-2细胞,分为对照组、白蛋白(BSA)组和白蛋白加抑制剂(BSA+Ly294002)组.分别于培养12、24、48 h后,用MTT法检测HK-2细胞的增殖;RT-PCR检测HK-2细胞TGF-β1 mRNA的表达;Western印迹测定HK-2细胞TGF-β1和磷酸化的PI3K(p-PI3K)、Akt(p-Akt)蛋白分泌.结果 白蛋白可明显诱导HK-2细胞增殖(P<0.05),诱导后12 hBSA组与对照组比较,TGF-β1 mRNA表达明显上调(0.472±0.025比0.233±0.021,P<0.05);TGF-β1蛋白明显上调(296±20.1比100±13.2,P<0.05);p-PI3K、p-Akt蛋白表达显著增加(P<0.05).加Ly294002 12 h时HK-2增殖受抑制;48 h时TGF-β1 mRNA、蛋白和p-PI3K、P-Akt蛋白的表达也受到抑制(均为P<0.05).结论 白蛋白通过活化PI3-K/Akt信号转导通路诱导肾小管上皮细胞产生TGF-β1.     

Silent information regulator 1 (SIRT1) promotes the migration and proliferation of endothelial progenitor cells through the PI3K/Akt/eNOS signaling pathway

Silent information regulator 1 (SIRT1) mediates many effects of caloric restriction (CR) on an organism’s lifespan and metabolic pathways. Recent reports have also emphasized its role in vascular function. The present study was designed to investigate the effects of SIRT1 on the properties of mouse spleen derived endothelial progenitor cells (EPCs). SIRT1 in EPCs was significantly increased by serum and by vascular endothelial growth factor (VEGF). Moreover, an adenovirus (Ad) vector expressing SIRT1 (Ad-SIRT1)-mediated overexpression of SIRT1 directly enhanced migration and proliferation of EPCs, whereas silencing of endogenous SIRT1 in EPCs inhibited cell functions. In addition, LY294002 (a PI3K inhibitor), sc-221226 (an Akt inhibitor), and L-NAME (an NOS inhibitor) abolished Ad-SIRT1-induced migration and proliferation of EPCs, and prevented nitric oxide (NO) production. Phosphorylation of Akt, PI3K, and endothelial nitricoxide synthase (eNOS) were up-regulated by Ad-SIRT1, which was attenuated by LY294002, sc-221226, and L-NAME. Together, the results suggested that through the PI3K/Akt/eNOS signaling pathway, SIRT1 plays an important role in the biological properties of EPCs.